Anopheles maculipennis, the historic vector of malaria in Europe and the Middle East was the first sibling species complex to be discovered among mosquitoes 12. The Maculipennis complex formally comprised 12 Palearctic members including Anopheles atroparvus, Anopheles beklemishevi, Anopheles labranchiae, Anopheles maculipennis, Anopheles martinus, Anopheles melanoon, Anopheles messeae, Anopheles sacharovi, Anopheles persiensis, An. daciae, An. lewisi and An. artemievi 34567. Anopheles sacharovi is the main vector in Turkey and is, together with Anopheles superpictus and Anopheles pulcherrimus, the most important vector, of malaria in the former Soviet Union, although An. messeae has been implicated in the resurgence of malaria in Russia and the Ukraine 8. Three species of the Maculipennis complex, An. atroparvus, An. labranchiae and An. sacharovi are known to be efficient current or historical vectors of malaria in the Palearctic region 910. An. maculipennis s. s. has been identified as the major vector of malaria on the Caspian Sea coast area of Iran and An. sacharovi is considered the main vector in the central plateau of the country 1112. Djadid 13 reported that members of this species complex in northern Iran are active from May to September, with a peak in July and that they breed readily in rice fields, spring and clean standing water, and adult mosquitoes could be found in animal shelters (95%). These species were susceptible to dieldrin, malathion, deltamethrin and resistant to DDT. Their biting pattern on human and animals baits is more or less the same, starting at 19.00 hrs with a peak between 20.00 – 21.00 hrs. Enzyme-linked immunosorbent assay (ELISA) carried out on 304 blood-fed mosquitoes collected from indoor resting sites revealed that they have fed predominantly on cattle, with fewer blood meals on sheep and poultry 13. None of the mosquitoes had fed on human blood although a previous study by Manouchehri et al. 14 has shown an anthropophilic index for this species in northern Iran of 1.7–4.9%. Furthermore, Djadid 13 reported that hibernation in this species starts in October and that complete fat body could be seen in February. The following culicidae mosquitoes larvae have been found in An. maculipennis breeding places; Anopheles hyrcanus, Anopheles claviger, Culex pipiens, Culex mimeticus, Culex tritaeniorhynchus, Aedes vexans, Culiseta subochra, Uranotaenia unguiculata. Adult of Anopheles algeriensis and Anopheles hyrcanus also has been found in resting places of An. maculipennis 13.

From an operational point of view in malaria control, in the region to the north of the Zargors range of mountains, An. maculipennis, An. sacharivi and An. superpictus are recognized as malaria vectors. This region was malaria-free for more than 30 years. However, since 1994, malaria has been re-introduced to this area through Republic of Azerbaijan and Armenia 1516.

Several studies have employed ecological, morphological, physiological and biochemical data to characterize members of the An. maculipennis complex including the preferences 17, larval chaetotaxy 18, ovarian polytene chromosome banding pattern 1920 cuticular hydrocarbons 21 isoenzyme analysis 2223 and most recently, DNA sequences 24252627282930. Of these methods, egg morphology 31 and DNA sequencing 73233 have been used for identification of different members of this complex in Iran.

Few studies have been carried out in northern areas of the country where malaria has reappeared and been introduced into several different provinces. Although, it has been postulated that members of An. maculipennis complex are responsible for malaria transmission, how many species within maculipennis complex are present in Caspian Sea region has not been defined yet. Do they exist as sympathric species and what if any, is, their role in malaria transmission? In view of the zoogeographical, ecological and social changes on both sides of Caspian Sea, this study was carried out in order to provide further evidence on the status of species composition and to identify more accurately the members of An. maculipennis complex in northern Iran. Field collection, morphological identification followed by amplification and sequencing of ITS2 region led to identification of six members of An. maculipennis complex and their comparison with those related sequences deposited in GenBank. The occurrence of three species of An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae is reported for the first time from northern Iran. This proves the extended distribution of these species towards southern territory of the An. maculipennis complex. This is a pre-requisite for understanding the epidemiology of malaria on both sides of Caspian Sea, and attempting to prevent the re-introduction of malaria to malaria-free areas.

The rDNA-ITS2 region was amplified by PCR from genomic DNA of 235 specimens from the five provinces of East Azerbaijan, Ardebil, Guilan, Mazandran and Khorassan in Iran (Figure 1). Sequences for the ITS2 region were obtained from 28 specimens, which have been deposited in GenBank (Table 1). The size of sequenced PCR amplified fragments in different geographical populations of the An. maculipennis complex varied from 453 bp in An. messeae to 498 bp in An. sacharovi, but with no intra-specific variation. Based on comparison of these sequences with the available ITS2 sequences of An. maculipennis complex in GenBank, six known species of the complex were identified in Iranian specimens including: An. maculipennis (100% identity with GenBank: AY365010 from Greece), An. sacharovi (100% identity with GenBank: AY533588 from Greece), An. persiensis (100% identity with GenBank: AY137819 from Iran), An. messeae (99% identity with GenBank: AY504236 from U.K.), An. atroparvus (99% identity with GenBank: AY365007 from Italy) and An. labranchiae (100% identity with GenBank: AY365008 from Italy). The sequence alignment of the six Anopheles species is shown in Figure 2. Presumptive boundaries of 5.8 s and 28 s genes were deduced from the comparison of alignments in the sequences of other mosquitoes 263741. The size of ITS2 sequences in six species ranged from 283 bp in An. maculipennis to 302 bp in An. sacharovi (Figure 2). The ITS2 region in all six species identified molecularly in this study started with

TTGACC

TTGATC

The overall average base composition was 24.89% (23.21–28.48%) for A, 22.36% (21.77–23.19%) T, 24.91% (23.43–25.58%0) G and 27.84% (25.83–28.91%) for C. Percentage of GC content was 53.35 in An. maculipennis (ITS2 = 283 bp), 49.33 in An. sacharovi (ITS2 = 302 bp), 51.74 in An. persiensis (ITS2 = 286 bp), 54.76 in An. atroparvus (ITS2 = 294 bp), 53.74 in An. messeae (ITS2 = 292 bp) and 53.58 in An. labranchiae (ITS2 = 293 bp). These values are concordant with 49.4–54.1% GC values reported for other Palearctic members of An. maculipennis complex 26274344.

The group of Anopheles mosquitoes referred to as the Maculipennis complex include the most important malaria vector of the Palearctic region, which is difficult or impossible to identify by their morphological characteristics 47. However, for the identification of two of the Nearctic An. maculipennis species, An. hermsi and An. freeborni, a PCR assay has already been established 42. Subsequently, Proft et al. 27 developed a diagnostic PCR system to differentiate between six of the seven An. maculipennis sibling species occurring in Europe including An. maculipennis, An. sacharovi, An. melanoon, An. atroparvus, An. labranchiae and An. messeae. Romi et al also designed a heteroduplex analysis based on the ITS2 sequence which enabled seven species of the complex including An. maculipennis, An. sacharovi, An. martinius, An. atroparvus, An. labranchiae, An. melanoon and An. messeae to be identified 47.

This study has been designed in order to provide molecular evidence and to verify the real composition of the An. maculipennis complex in northern Iran. The mosquito fauna in Iran has not been extensively studied since 1986 48 and later when Anophelines larvae were studied by Saebi 49. However, regarding the An. maculipennis species complex, Faghih et al. 50 and Manouchehri et al. 14 claimed the presence of four species including An. maculipennis s.s. (typicus) in Ramsar (Mazandran province), Anopheles subalpinus in Sari, Babolsar, Chalous (Mazandran province), Astaneh (Guilan province), An. melanoon in Astaneh (Guilan) with An. subalpinus and An. melanoon sympathric in Astaneh and on the border of the two provinces of Guilan and Mazandran in northern Iran. They reported the presence of An. sacharovi in all areas of these two provinces. Djadid 32, working on An. maculipennis, An. sacharovi from Iran, An. beklemishevi and two cytogenetically different forms of An. messeae from Russia has found three species of An. sacharovi [GenBank: AY842517], An. messeae [GenBank: AY050639], and An. atroparvus [GenBank: AY050640] in Iran by using RAPD, SSR and ITS2 sequences. Recently, Sedaghat et al. 751 reported the presence of three genetically distinct species of the An. maculipennis complex from Iran, including An. maculipennis, An. sacharovi, and An. persiensis. The sequence of An. persiensis was identified in 2002 by Djadid and Romi from Rasht (Guilan province) and Amol city in Mazandran province, and indeed it was described later by Sedaghat et al. 7. This species has been found only in the northern Caspian Sea littoral provinces of Guilan and Mazandran. Oshaghi et al. 33, working on members of An. maculipennis complex from the north-west and central regions of Iran, only found An. maculipennis and An. sacharovi. However, in study, for the first time, the presence of six species of this complex (An. maculipennis, An. sacharovi, An. persiensis, An. atroparvus, An. labranchiae and An. messeae) are reported in northern Iran based on the sequence of rDNA-ITS2. The three species of An. messeae, An. atroparvus and An. labranchiae have not been reported before in Iran.

The ITS2 sequences of six Palearctic species of An. maculipennis complex varied in length from 283 bp in An. maculipennis up to 302 bp in An. sacharovi. This is in the range of ITS2 length in other examined Anopheles species; 363–369 bp in Anopheles nunestovari 52, 287–329 bp in Anopheles quadrimaculatus complex 53, in the North American species of the An. maculipennis complex 305 – 310 bp 42 and in seven Palearctic members of An. maculipennis complex about 280–300 bp 26. However, the sequence of each species from Iran and other parts of the world within this complex is highly conserved, with about 99–100% similarity.

In a previous study by Marinucci et al. 26, the phylogenetic relationships among the members of the An. maculipennis complex inferred by maximum parsimony analysis of the PAUP programme and neighbour joining and maximum likelihood analysis of the PHYLIP program. All the trees obtained were almost identical in topology although the relationships among the three species i.e. An. maculipennis, An. messeae and An. melanoon, remained unresolved. Perhaps due to the differentiation of these species from neighbouring taxa within a brief evolutionary time-frame that dispensed insufficient differences to support these individuals' lineages 26. Recently rDNA-ITS2 sequences of three other Palearctic members of the An. maculipennis complex have been reported from Romania and Russia, namely An. daciae, An. artemievi and An. beklemishevi 29304546. Kampen analysed An. beklemishevi specimens from Russia by their ITS2 ribosomal DNA sequences to amend and to specify the phylogenetic tree of the An. maculipennis species complex 30. The results generally correspond with the data presented in the current study except that the constructed trees do not include An. persinsis, and that An. messeae and An. atroparvus are not as close as demonstrated by Kampen 30. He showed that An. beklemishevi is in a closer relationship to the Nearctic rather than to the Palearctic sibling species, which is in concordance with the demonstration of final phylogenetic tree drawn from this study by including An. beklemishevi sequence (GenBank: AY593958) to other sequences (Figure 4). However, An. sacharovi as a Palearctic member of An. maculipennis complex perhaps due to its common evolutionary speciation, seems to be the closest taxa to both the Nearctic species and An. beklemishevi.

The phylogenetic tree generated in the current study has separated Nearctic members of An. maculipennis complex into two distinct lineages (Figure 3). In agreement with Marrinucci et al. 26, An. occidentalis was placed in a sister group in relation to An. hermsi-An. freeborni. An. sacharovi placed in a sister group to the remainder of the Palearctic group. The An. labranchiae-An. atroparvus clade was the sister group to An. maculipennis, An. persiensis and An. messeae. However, interestingly, the newly described member of complex, An. perciensis is closer to An. sacharovi, perhaps revealing its common evolutionary background with this species as compared to other members of An. maculipennis (Figures 3 and 4).

With regards to the presence of six members of An. maculipennis complex, further complementary ecological studies are needed in order to determine the role of each species in malaria transmission in different areas of northern Iran. To achieve this goal, some field experiments were carried out. The preliminary results of dissection of An. sacharovi (species identification confirmed by ITS2 sequence analysis) collected during the suspected hibernation period (October-March) showed that this species will go into hibernation with gonotrophic dissociation. In this case, all dissected mosquitoes have shown no dilatation proving that the female adult mosquitoes over winter as nuliparus and the last blood they took will be used for producing the fat body allowing the female to start the next generation in the beginning of next seasonal activity. However, it remains un-clear how these ecological data fit with the presence of one or more species within An. maculipennis complex in northern Iran, and what is the role of other anophelines in this region (i.e. An. superpictus, An. hyrcanus, An. claviger, An. algeriensis, Anopheles pseudopictus) in malaria transmission and since re-introduction of malaria in northern Iran.

Beklemishev 54 listed eight major factors that determine epidemiological efficacy of malaria vectors in Russia: susceptibility of mosquitoes to Plasmodium parasites, sporozoite survival in salivary glands, female feeding behavior, absolute and relative number of mosquitoes, seasonal dynamics of mosquito densities, survival rate and infective period of mosquito females, ambient temperature and winter diapauses of adult females in a state of gonotrophic dissociation. Besides previous studies indicated that the sibling species are not equally important as vectors for malaria parasites because of their feeding preferences and their differential susceptibility to infection, as described in complex species of Anopheles culicifacies, Anopheles gambiae, Anopheles fluviatilis 105556575859. This may be grounds for re-considering the importance of other previously identified species in this region and their role in malaria transmission.

Nowadays, the most important vectors are considered to be An. sacharovi, which is responsible for the majority of Plasmodium vivax transmissions in the Asian part of Turkey 60, and An. labranchiae, formerly was the main vector of malaria in Italy 9. An. atroparvus is the most efficient vector in Britain 43. An. maculipennis, An. messeae and An. sacharovi are capable of transmitting malaria; however, they exhibit different vector capacities 86061. In this regard, detection of three species of An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae, as new records in northern Iran, is a case for concern because of their potential in malaria transmission and more important, the extent of their geographical distribution towards southern territory of the An. maculipennis complex.

A better understanding on the epidemiology of malaria on both sides of the Caspian Sea may be provided by applying the molecular techniques to enable the correct identification of species complexes, the detection of plasmodium composition in anopheles vectors and the status of insecticide resistance by looking at related genes. In addition, it is worth remembering that as the An. maculipennis complex is distributed over an area that covers central Iran to the Caspian Sea region and up to Sweden, an international effort is required to prevent the re-introduction of malaria to those areas where "anophelism without malaria" prevails.